RNA bacteriophage-based delivery system

ABSTRACT

A delivery system, especially for delivery to targeted sites in the human or animal body, comprises capsids of the coat protein amino acid sequence of phage MS-2 or related phage, or a modification thereof which retains capsid-forming capability, and at least some of the capsids enclosing a moiety foreign to the genome of MS-2 or related phage.

This invention relates to a protein-based delivery system and isparticularly directed to the delivery of encapsidated foreign moieties,especially to targeted sites in the human or animal body.

There is increasing interest in the targeting of foreign moieties to thesites in the body where their activity is required. Thus it is importantthat drugs, particularly those having undesirable side effects, aredelivered to the site where they are to act. Many other molecularspecies require to be delivered in a site specific manner, often toparticular cells, for example polynucleotides (anti-sense or ribozymes),metabolic co-factors or imaging agents. One such system has beendescribed by Wu et al., J. Biol. Chem., 263, 14621-14624 andWO-A-9206180, in which a nucleic acid useful for gene therapy iscomplexed with polylysine linked to galactose which is recognised by theasialoglycoprotein receptors on the surface of cells to be targeted.However, there are many occasions, such as in the delivery of acytotoxic drug, when it would not be satisfactory to use a deliverysystem in which the moiety to be delivered is so exposed. There istherefore a need to develop alternative delivery systems which have theflexibility to target a wide range of biologically active foreignmoieties.

Co-pending UK patent applications no. 9114003.8 and 9201372.1 describethe modification of the coat protein of phage MS-2 as a presentationsystem for epitopic species, which may be included in a modified coatprotein sequence or attached to the coat protein via a cysteine residueand optional further spacer. These applications relied on the ability ofthe coat protein of MS-2 and similar phages to be cloned and expressedin a bacterial host such as E. coli as largely RNA-free empty phageparticles. Romaniuk et al., (1987), Biochemistry 26, 1563-1568 havestudied the relationship between the MS-2 coat protein and the RNAgenome. It is apparent that, although RNA-free coat protein assembliescan be produced in E. coli, capsid formation in natural infections istriggered by coat protein interaction with a 19 base stem-loop(translational operator) in the RNA genome sequence. Talbot et al.,1990, Nucleic Acids Research 18, No. 12, 3521-3528 have synthesised the19 base sequence and variations of this sequence and investigated therecognition and binding by the coat protein. It has been found that notonly does the translational operator RNA signal exist as the stem-loopstructure within the larger genomic RNA but that it is also recognisedas the short fragment of just 19 bases. This fragment has the ability tocause recombinant coat protein to bind specifically and self-assemblearound it, resulting in recombinant capsids containing multiple copiesof the RNA fragment.

According to the present invention there is provided a delivery systemcomprising capsids of the coat protein amino acid sequence of phage MS-2or related phage, or a modification thereof which retains capsid-formingcapability, or sufficient of said sequence or modification to retaincapsid-forming capability, at least some of said capsids enclosing amoiety foreign to the genome of MS-2 or related phage.

The foreign moiety is suitably attached to a portion of the RNA genomesequence of MS-2 or related phage capable of functioning as atranslational operator for capsid formation, or a variant thereofretaining the translational operator function. The RNA genome sequencewas first defined by Fiers, Nature, 1976, 260, 500-517, and we havefound that the 19-base stem loop (bases -15 to +4 relative to the startof the replicase gene) SEQ ID NO. 1 or a variant thereof, especially thevariant where cytidine is substituted at the -5 position, is the minimumrequirement for function as the translational operator (see Talbot etal., 1990, Nucleic Acids Research 18, No. 12, 3521-3528). The foreignmoiety may be attached directly to the operator sequence or via a spacermoiety, for example a series of uridine residues (suitably 6) to ensurethat the foreign moiety does not interfere with the operator function.

According to a preferred form of the invention the coat protein aminoacid sequence has been modified to provide a site suitable forattachment thereto of a targeting moiety. The invention includes capsidshaving such a site for subsequent attachment of a targeting moiety andcapsids to which the targeting moiety has already been attached.

The coat protein amino acid sequence is preferably that derived fromphage MS-2, but it may also be derived from related RNA-phages capableof replication in E. coli, such as phages R17, fr, GA, Qβ and SP. SuchRNA-phages of physical structure similar to that of MS-2 will containsome chemical variation in the amino acid residues of the coat proteinand are thus conservatively modified variants of MS-2 coat protein.While it is believed at present that substantially the entire coatprotein may be required for capsid assembly, deletions and/or insertionsare also possible whilst still retaining capsid-forming capability.Proteins having such modified sequences are included within the scope ofthe invention.

The three-dimensional structure of the MS-2 phage particle has beenpublished by Valegard et al., (Nature, 1990, 345, 36-41). The publisheddata show that, firstly, the structure of the coat protein is notrelated to the eight-stranded β-barrel motif found in all otherspherical RNA virus subunits whose structures are known at the presenttime. Secondly, although the coat protein exhibits quasi-equivalentinter-subunit contacts, there are no other devices, such as extendedarms of polypeptide, helping to secure each protein conformer. The coatprotein structure can be viewed in terms of three separate regions.These are not domains in the usual sense but could represent independentfolding units. These regions are residues 1-20, which form the β-hairpinstructure which protrudes from the surface of the phage forming the mostdistal radial feature. This region is followed by residues 21-94 whichform five β-strands including the "FG-loop" which is the site of theonly major conformational change between quasi-equivalent conformers.These β-strands are then followed by two α-helices, residues 95-125,which interdigitate to secure dimers of the coat protein sub-units.Valegard et al. are concerned solely with the physical structure of theMS-2 virus and do not attempt to elucidate the mode of action of thevirus.

Co-pending UK patent application No. 9114003.8 describes theintroduction of a cysteine residue into the N-terminal protruberantβ-hairpin of the coat protein (with removal of the cysteine residuespresent externally of the N-terminal protruberant β-hairpin). Such acysteine residue provides a preferred site for attachment thereto of atargeting moiety. The resultant coat protein has therefore been somodified in the region of amino acid residues 1 to 20, such numberingbeing with reference to the entire coat protein sequence of MS-2 aspublished by Fiers, Nature, 1976, 260, 500-507. Preferably themodification to introduce the cysteine residue is towards or at themiddle of the hairpin region. It is preferred to introduce the cysteinein the region of the glycine 13 and 14 residues of the coat protein. Thecysteine residues to be removed which are external of the β-hairpin arefound at positions 46 and 101. They may be removed by any convenientconventional genetic engineering technique, suitably by site-specificmutagenesis.

In a preferred method of removing the unwanted cysteine residues, twomutants of the MS-2 coat protein, one singly mutated at cys 46 and onesingly mutated at cys 101 may be obtained by standard commerciallyavailable techniques for site specific mutagenesis and the correspondingcDNA sequences introduced into standard expression vectors, whichvectors are subjected to digestion with restriction enzymes to obtainseparately the DNA fragment containing the mutated cys 46 site and thecorresponding fragment containing the mutated cys 101 site, thefragments being subsequently ligated to give a doubly-mutated coatprotein cDNA. The doubly-mutated cDNA may then be subjected tosite-directed mutagenesis using standard methods to introduce a cysteineresidue in the β-hairpin region.

Alternative modification of the coat protein which enables targeting ofthe encapsidated moiety may include insertion of peptide sequences inthe protruberant β-hairpin of the MS-2 coat protein as described inco-pending UK patent application no. 9201372.1.

The cysteine residue, or alternative modification site, can be furtherlinked to a targeting moiety with or without interposition of a furtherspacer moiety. An example of such a targeting moiety is a galactoseresidue which can be used to direct the capsids to interact withspecific cell surface receptors and thus carry the foreign moiety withinthe capsids to and/or into specific cells. Other possible targetingmoieties are other cell surface receptor ligands or monoclonalantibodies. Suitable receptors for the targeting moieties are theasialoglycoprotein receptor and the receptor for melanocyte stimulatinghormone.

Suitable spacer moieties, if employed, are selected from knowncommercially available heterobifunctional crosslinking reagents whichcouple with the exposed cysteine thiol group. Examples of suchcross-linkers are m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-succinimidyl-(4-iodoacetyl)aminobenzoate andN-succinimidyl-3-(2-pyridyldithio)propionate. The choice of crosslinkerwill depend on the targeting moiety and its size. Thus larger molecularspecies may require longer crosslinking moieties to minimise sterichindrance. The crosslinker may be linked first to the cysteine residueor first to the targeting moiety.

Alternatively the thiol function (or other derivatisable group) can beintroduced into wild type, empty capsids of MS2 coat protein usingsuitable heterobifunctional chemical reagents such as N-succinimidylS-acetylthioacetate (SATA).

The foreign moiety held within the capsids can vary widely and includegenes and gene fragments, ribozymes, anti-sense messages or cytotoxicand chemotherapeutic agents intended for such purposes as anti-sensegene therapy or selective killing of target cells.

The form in which the foreign moiety is held within the capsids willdepend on the release properties required. For release at the targetedsite it will be important to ensure that the right conditions prevail,for example to permit cell localisation and internalisation via receptormediated endocytosis.

The capsids may suitably be obtained by first obtaining empty MS-2capsids, for example by expression of vectors containing coat proteincDNA in E. coli as described in co-pending UK application No. 9201372.1.The MS-2 capsids may be of wild type MS-2 coat protein or have beenmodified, for example to introduce a cysteine site as described inco-pending UK application No. 9114003.8. The capsids are thendisassembled, for example, at acid pH (e.g. using acetic acid), beforereassembly suitably at raised pH, e.g. pH 7. In the presence of thedesired foreign moiety linked to an RNA sequence capable of functioningas the translational operator in the reassembly of the coat proteinaround the RNA sequence and foreign moiety. Other methods of disassemblymay be used, for example in the presence of urea. It is alsocontemplated that the capsids enclosing the foreign moiety may beobtained by random incorporation of the moiety in the capsids.

The RNA sequence may be obtained by biochemical methods from thecomplete MS-2 RNA genome. Alternatively, the RNA sequence is obtained bychemical synthesis, for example as described by Usman et al., (1987), J.Am. Chem. Soc., 109, 7845-7854. Chemical synthesis is preferred as itenables ready addition of any spacer moiety and linking to the foreignmoiety to be delivered.

It will be apparent that there are several advantages in using MS-2 andrelated phages as a presentation system. Thus the empty coat proteincapsids can be readily expressed in comparatively high yield in E. coliand the product is easily purified (see R. A. Mastico et al. J. Gen.Virol. (1993) 74, 541-548 the contents of which are herein incorporatedby reference). It has been found that the assembled capsids showconsiderable stability with respect to a range of temperatures, pH andionic strength.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by the accompanying drawings, inwhich:

FIG. 1 represents a trace obtained from an HPLC gel filtration columndescribed in Example C, when wild type MS-2 capsids which had not beendisassembled and reassembled were used;

FIG. 2 represents a trace obtained from an HPLC gel filtration columnusing molecular weight standards;

FIG. 3 represents a trace obtained from an HPLC gel filtration columndescribed in Example C, when mixture (i) is used;

FIG. 4 represents a trace obtained from an HPLC gel filtration columndescribed in Example C, when mixture (ii) is used;

FIG. 5 represents a trace obtained from an HPLC gel filtration columndescribed in Example C, when mixture (iii) is used;

FIG. 6 represents a trace obtained from an HPLC gel filtration columndescribed in Example C, when mixture (iv) is used;

FIG. 7 is a graph representing the results obtained from the experimentdescribed in Example C;

FIG. 8 is an SDS-Page gel of cys 14 modified MS-2 capsid;

FIG. 9 is an electromicrograph of the immunoaffinity purified cys 14modified MS-2 capsid;

FIG. 10 shows a control curve for reaction of free cysteine with DTNB,as described in Example E, on page 15;

FIG. 11 shows the curve obtained when cysteine was mixed with 1 to 10 mMactivated galactose and left to stand at room temperature for 1 hour,followed by assay using DTNB, as described in Example E;

FIG. 12 shows macrophage sections negatively stained, as described inExample H;

FIG. 13 shows macrophage sections negatively stained, as described inExample H;

FIG. 14 shows macrophage sections treated with anti-rabbit peroxidase,which then stains the anti-MS-2 antibodies, as described in Example H;and

FIG. 15 shows macrophage sections treated with anti-rabbit peroxidase,which then stains the anti-MS-2 antibodies, as described in Example H.

The invention will now be described by way of example.

A) Preparation of MS-2 Coat Protein Capsids

The coat protein of MS-2 was obtained by growing phage MS-2, purifyingthe RNA, followed by oligonucleotide primer directed reversetranscription to produce single-stranded cDNA which was converted todouble stranded cDNA using oligo primers and Klenow polymerase. The cDNAwas then subcloned into an expression vector pGLWll (Smith, M. C. M.,Czaplewski, L. G., North, A. K., Baumberg, S. and Stockley, P. G. (1989)Mol. Microbiol. 3, 23-28,) placing the coat protein under the control ofthe inducible tac promoter.

The pGLWll expression vector was expressed in E. coli and the cellularproteins obtained, purified and characterised as follows:

Standard laboratory strains of E. coli were transformed (to ampicillinresistance) with the expression plasmid carrying the recombinant MS-2coat protein gene. Rapidly growing cultures of these transformants inrich media were induced by addition of isopropyl-β-thiogalactoside(IPTG) to a final concentration of 1 mM when the O.D.₆₀₀ of the culturewas between 0.4-0.6. Cell growth was continued overnight before thecells were harvested by centrifugation, resuspended in neutral buffer,sonicated to lyse the cells, followed by centrifugation to separate thesupernatant (containing the expressed recombinant products) and cellulardebris. The supernatant was fractionated by ammonium sulphateprecipitation, the pellet of product being resuspended in buffer beforebeing purified on the basis of size by either sucrose density gradientsor gel filtration chromatography or by immuno-affinity chromatography(Mastico et al. (1993)). The product was obtained in the form of capsidswhich were subsequently disassembled by the addition of 2 volumes ofglacial acetic acid.

B) Preparation of RNA Genome Oligonucleotides

Four oligonucleotides were prepared by solid phase chemical synthesisusing 2'-silyl-protected phosphoramidite starting materials as describedby Usman et al., (1987), J. Am. Chem. Soc., 109, 7845-7854 and Talbot etal., (1990), Nucleic Acids Research, 18, No. 12, 3521-3528.

a) Containing nucleotides -15 to +4 of the RNA genome of MS-2 with acytidine introduced at position -5, i.e. a sequence encompassing thetranslational operator (described hereinafter as "MS-2C").

b) MS-2C carrying a 5' biotin residue as a model foreign moiety;

c) As for a), but with 6 uridine residues linked at the 5' end of theoligonucleotide (described hereinafter as "MS-2C+6U") also carrying as amodel foreign moiety a 5' biotin residue.

d) As for a) but with a 3' extension of 22 deoxynucleotides which arecomplementary, i.e. anti-sense, to the first 22 nucleotides of the mRNAfor the HIV-1 Tat protein.

The biotin group in b) and c) was introduced as follows:

500 mg of "DMT biotin-C6-PA" (a dimethoxytrityl-protectedbiotin-C6-spacer-phosphoramidate reagent available from CambridgeResearch Biochemicals Ltd., Cheshire, UK) was dissolved in 0.6 ml ofanhydrous acetonitrile. The synthesis of both MS-2C+biotin andMS-2C+6U+biotin was carried out on an Applied Biosystems Model 391 DNAsynthesiser on a 1 μMol scale.

Results of the syntheses with biotin:

    ______________________________________                                                              Average                                                              Overall Yield                                                                          Stepwise Yield                                          ______________________________________                                        MS-2C + biotin 62.4%      97.5%                                               MS-2C + 6U + biotin                                                                          38.9%      96.2%                                               ______________________________________                                    

The solid supports from all four syntheses were transferred into cleanvials and treated with HPLC grade methanol saturated with ammonia for 24hours at room temperature. The supernatants were transferred to freshvials and dried down using a stream of nitrogen gas.

The resulting pellets were then resuspended in t-butylammonium fluoridein THF, and incubated at room temperature for 24 hours, and thenquenched using an equal volume of 1M ammonium acetate.

The quenched deprotection reactions were then desalted using PharmaciaNAP 25 columns. The columns were equilibrated with 25 mls 0.2× TBE and2.0 mls of sample was added to the columns, with 0.5 ml fractions beingcollected. Once all the material had been eluted, the columns werere-equilibrated as before and the second half of the sample was desaltedlikewise. A fresh column was used for each RNA oligonucleotide.

The size of each product was confirmed by 3' radiolabelling andchromatography over polyacrylamide sequencing gels. Auto radiographyrecorded single dominant radioactive species with expected mobilities.

C) Reassembly of Capsids in the Presence of Oligonucleotide-Biotin andMS-2C-Anti-Tat

Reassembly of the disassembled MS-2 capsids described in A) above wascarried out by raising the pH from 2.4 to 7.0 in the followingcircumstances and in each case in the presence of an avidin-fluoresceincomplex (Pierce Europe BV, Holland).

i) MS-2 coat protein only (no oligonucleotide present)

ii) MS-2 coat protein plus MS-2C

iii) MS-2 coat protein plus MS-2C+6U-biotin

iv) MS-2 coat protein plus MS-2C-biotin.

The resulting capsids were separated on an HPLC gel filtration columnand the results are shown in FIGS. 1 to 6 where FIG. 1 represents thetrace obtained using wild type MS-2 capsids which had not beendisassembled and reassembled and FIG. 2 represents molecular weightstandards. The peak eluting after approximately 19 minutes correspondedto the assembled MS-2 capsids. FIGS. 3 to 6 represent the tracesobtained for the mixtures i), ii), iii) and iv) respectively. It will beseen that while i) shows only a small indication of capsid formation,ii) shows a greatly increased amount of capsid as expected while iii)and iv) show retained capsid formation in the presence ofavidin-fluorescein which will complex with the biotinylatedoligonucleotides.

The presence of the avidin-fluorescein-biotin complex within the capsidswas demonstrated by measuring the fluorescence intensity (resulting fromfluorescein) in the fractions corresponding to the capsid peaks in FIGS.3, 4 and 5 (i.e. for i), ii) and iii) above) plus additionally amixture:

v) MS-2 coat protein plus 1:1 mixture of MS-2C+6U-biotin and MS-2C (nobiotin) plus avidin-fluorescein complex.

The results are shown in FIG. 7 and it will be seen that iii) showed thepresence of fluorescence in the fractions corresponding to the capsidswhile v) showed the presence of a reduced level of fluorescence when thebiotinylated oligonucleotide was diluted 1:1 with non-biotinylatedoligonucleotide. The avidin-biotin complex is thus shown as being withinthe capsids.

MST WT capsids are reassembled with the (MS-2C-anti-Tat) as follows:

a) Reassembly

MS2 WT CP was purified as usual, but concentrated to 10 mg/ml byspinning down at 35 k rpm for 6 h at 4° C. 0.4 ml of the MS2 (10 mg/ml)was added to 0.8 ml of glacial acetic acid and kept on ice for 30 min.The precipitates were removed by centrifugation at 6500 rpm, 4° C. for20 min and the supernatant passed over a NAP-25 column equilibrated with1 mM acetic acid. Both the protein and the oligonucleotide were mixedwith 10× TMK buffer (100 mM Tris, 80 mM KCl and 10 mM MgCl₂) and kept onice for 1 h. The MS2 CP was added to the MS-2C-anti-TAT solution atmolecular ratios from 180:1 to 2:1 and the mixture incubated at 37° C.for 1 h and then RT for 6 h. The reassembled capsids were stable at 4°C. for 2 weeks.

b) Analysis of the Reassembled Capsids

In order to confirm that the reassembled particles had encapsidated thetest oligonucleotide capsids were separated from other components byHPLC gel filtration chromatography as described above. The peakcorresponding to capsids was then phenol extracted, the nucleic acidsprecipitated with ethanol, the precipitate radiolabelled with ³² P andelectrophoresed over a denaturing polyacrylamide gel. The results showedthat a nucleic acid fragment with identical mobility to the startingmaterial had been recovered from the HPLC column confirmingencapsidation. Finally, we used transmission electron microscopy (asdescribed above) to investigate the reassembled particles. This showedthat the bulk of the input coat protein had reassembled into capsids ofsimilar size and symmetry to the wild type phage.

C) Eukaryotic Cell Transfection

Human Hela cells were grown in Dulbecco modified Eagle mediumsupplemented with 10% fetal calf serum. Twenty four hours beforetransfection the cells were plated out at 1×10⁶ cells per 75 cm² flask.The medium was changed half an hour before the transfection.

The cells were transfected with 5 μg LTR Cat and 5 μg pSVTat (1) in 0.5ml of calcium phosphate-DNA coprecipitate (2). After 16 hours the cellswere washed with PBS and fresh medium was added. The cells were thenincubated for 24 hours before harvesting. The transactivation of LTRCatby Tat was challenged with the antisense oligo directed against thefirst 22 bases of the Tat mRNA. This was attached to the 3' end of theMS2 RNA stem loop (-15 to +4) to direct the reassembly of the capsidaround the oligo.

    (5'-ACA-UGA-GGA-UUA-CCC-AUG-U--TAC-CTC-GGT-CAT-CTA-GGA-TTG-3')                                                      SEQ ID NO:2                         

D) Preparation of Cysteine or Thiol-Modified Coat Protein Capsids

Cystene modified MS-2 coat protein was produced as follows:

Site directed mutagenesis and standard techniques were used to produceamino acid mutants at coat protein positions 46 and 101.

Mutants were selected having either cysteine at position 46 substitutedby serine or cysteine at position 101 substituted by serine.

Each single mutant DNA was expressed in E. coli to demonstrate theability to self assemble.

The ser 46 single mutant from step A) was introduced into standard coatprotein expression vector ptacACP and digested with SacI and XbaIrestriction enzymes and the longer backbone fragment so obtained treatedwith calf intestinal phosphatase and then purified on agarose orpolyacrylamide gels before electroelution and precipitation.

The ser 101 mutant was treated likewise with omission of the phosphatasetreatment. The smaller fragment containing the C-terminal portion of thecoat protein gene was purified by gel electrophoresis.

The large fragment containing the mutated cys 46 site and the smallfragment containing a mutated 101 site were ligated by standard methods.The recombinant molecules thus obtained were used to transform E. coliTGl to ampicillin-resistance and positive colonies checked for doublemutation by DNA sequencing.

The doubly-mutant ser 46/101 coat protein cDNA from step B wasintroduced into an M 13 sequencing vector by standard subcloningmethods, a single stranded template for site-directed mutagenesisgenerated and a cysteine residue introduced at gly 14 using thecommercially available site specific mutagenesis protocol based onnucleotide phosphothioates. There was thus obtained mutated cys 14 Ser46/101 coat protein cDNA.

The isolated mutated cDNA was expressed in E. coli to confirm thecapsid-forming ability of the recombinant protein. The cys 14 ser 46/101coat protein cDNA of C) above was introduced to expression vectorpTAC-CP and the resultant plasmid used to transform E. coli strain TGlin accordance with standard procedure. The cys 14 ser 46/101 coatprotein was then produced according to the following protocol.

5×5 ml (2TY media with 100 μg/ml ampicillin) cultures of single coloniespicked from transformation plates were grown for approx. 4 hrs at 37° C.and then used to inoculate 5×500 ml flasks of 2TY plus ampicillin andthe cultures were grown at 30° C. When the cultures reached OD₆₀₀approx. 0.45 protein production was induced by adding 1 mM IPTG. Cellsgrown overnight were then centrifuged at 3 k rpm, 30 mins, 4° C. in aBeckman JA14 rotor.

The resulting pellets were resuspended in 50 mM Hepes, 100 mM NaCl, 10mM dithiothreitol (DTT), 5 mM EDTA and 1 mM phenylmethyl sulphonylfluoride (PMSF), and the cells lysed by sonication. The cell lysate wasthen centrifuged at 15 k, 20 mins, 4° C. in a Beckman JA20 rotor and thesupernatant passed down a NAP-25 column (Pharmacia) to change buffers to20 mM NaPi (sodium phosphate-based buffer) pH 7.0. 1 ml fractions werecollected from the NAP column, the MS-2 coat protein containingfractions (nos. 2 to 5 inclusive) added to an anti-MS-2 coat proteinimmunoaffinity column and the sample allowed to bind for 1 hour at roomtemperature with gentle agitation.

The column was washed with 20 mM NaPi pH 7, then 10 mM NaPi/100 mM NaClpH 7. The sample was eluted with 20 mls 20 mM acetic acid/200 mM NaClapprox. pH 2.7 and the first 4 mls collected.

The pH was immediately adjusted by titration with 1M Tris.HCl pH 9 to pH7-7.4 and the mixture centrifuged at 30 k rpm, 4° C. overnight (approx.15 hrs) using a Beckman SW.55Ti rotor. The supernatant was decanted andthe MS-2 protein pellet resuspended in a small volume of the requiredbuffer.

Homogenous cys 14 modified capsids were obtained which were tested fortheir ability to react with an activated galactose reagent as describedin E) below.

SDS-PAGE of the resultant immunoaffinity purified cys 14 modifiedcapsids showed essentially a single component of the expected molecularweight. This result is shown in FIG. 8 where lane a) shows the cys 14modified capsids, lanes b) and c) show wild type capsids respectivelyimmunoaffinity purified and sucrose density purified and lane d) givesmolecular weight standards.

FIG. 9 shows an electromicrograph of the immunoaffinity purified cys 14modified capsids showing the presence of assembled particles similar tothose produced by wild type coat proteins.

Thiol groups can also be introduced into wild type assembled capsidsusing heterobifunctional reagents such as SATA, as follows:

2 mg of N-succinimidyl S-acetylthioacetate (SATA, PierceImmunotechnology) was completely dissolved in 0.5 ml ofdimethylformamide (DMF) by shaking, and 20 μl aliquots stored at 4° C.The purified MS2 WT CP was passed over a NAP-25 column equilibrated with0.05M phosphate buffer (PB,pH7.5) immediately before conjugation. 1 mlof MS2 CP (1 mg/ml) was mixed with 20 μl of SATA and kept at RT for 1 h(SATA:MS2=50:1). The solution was then deacetylated with 0.1 ml offreshly prepared hydroxylamine-HCl (25 mg in 0.5 ml H₂ O) at RT for 2 h.The MS2 derivative was separated from the reagent and by-products bydesalting over a NAP-25 column equilibrated in 0.1M PB pH7.5.

The stoichiometry of SATA groups introduced into wild type MS2 coatprotein was determined either by modification with DTNB (as describedbelow) or ³ H-iodo-acetic acid according to standard methods. Theresults suggest that approximately two new thiols per CP monomer areintroduced by the SATA modification. Both new thiols were completelymodified by treatment with activated galactose, as described below.

E) Reaction of Cysteine or SATA Modified Protein With ActivatedGalactose

In order to test the reactivity of the cys 14 modified MS-2 capsids, ahalogen-activated galactose was prepared as follows:

To a stirred solution of p-aminophenyl β-D-galactopyranoside (0.54 g; 2mmole) in water (4 ml) and ethanol (6 ml) was added iodoacetic anhydride(0.9 g; 2.5 mmole) at room temperature. After 2 hours, the reaction wasconcentrated to dryness and the residue washed with ether (2×10 ml).Crystallisation from ethanol gave the product as needles (0.7 g; 80%),mp 158-160° C.

The reaction of the cys 14 modified capsids with the activated galactosewas assayed using Ellman's reagent (dithionitrobenzoate, DTNB) whichgives a characteristic absorption at OD₄₁₂ on reaction with free --SHgroups. FIG. 10 shows a control curve for reaction of free cysteine withDTNB. The control curve was obtained using:

100 μl sample.

100 μl DTNB 4 mg/ml in 100 mM Na₂ HPO₄ pH8 ("buffer 1").

5 ml "buffer 1".

The mixture was left at room temperature for 15 minutes after addingDTNB and then the OD₄₁₂ recorded.

FIG. 11 shows the curve obtained when cysteine (4 mM) was mixed withfrom 1 to 10 mM activated galactose and left to stand at roomtemperature for 1 hour, followed by assay of these 100 μl aliquots asdescribed above using DTNB.

The cys 14 modified coat protein was reacted with DTNB as follows:

Purified cys 14 modified capsids were resuspended in buffer 1 containing1 mM EDTA to a final concentration of 400 μg/ml. The followingindividual experiments were set up.

1) 100 μl protein sample, 100 μl 5 mM activated galactose.

2) 100 μl protein sample, 90 μl buffer 1, 10 μl 5 mM activatedgalactose.

3) 100 μl protein sample, 100 μl buffer 1.

Each was left stirring at room temperature for 1 hour before addition of200 μl DTNB 4 mg/ml in ethanol to the stirring solution. OD₄₁₂ wasrecorded after 15 minutes and the results are shown in Table 1 below.The number of free thiols decreased with increasing exposure to thegalactose reagent, confirming that the cys 14 capsids had beenderivatised with galactose. Similar results were obtained withSATA--modified wild-type capsids.

                  TABLE 1                                                         ______________________________________                                        Sample                 O.D..sub.412 nm                                        ______________________________________                                              Buffer blank         0.0                                                1)    100 μl MS2-cys; 100 μl gal                                                                   0.031                                              2)    100 μl MS2-cys; 90 μl buffer; 10 μl gal                                                   0.080                                              3)    100 μl MS2-cys; 100 μl buffer                                                                0.118                                              ______________________________________                                    

F) Linking of cys 14 Modified Coat Protein to Immunogenic Peptide

The purified cys 14 modified capsids were linked as described below toHA10, a 10-mer peptide sequence encompassing a nonapeptide epitopederived from the haemagglutinin of the human pathogen influenza virusand having an N-terminal cysteine residue extension, which 9-mersequence YPYDVPDYA (SEQ ID NO:3) has been identified as containing oneof the antigenic determinants by Wilson et al., Molecular and CellBiology, May 1988, 2159-2165 and Cell, 37, 1984, 767-778. The procedureinvolved an initial crosslinking step to form a disulphide linkage whichwas then oxidised.

The following reagents were employed to make up four test reactionmixtures:

2 μg cys 14 modified capsids (about 3 μl ) ("cys bridge")

1 μl 1M Tris.HCl pH8, 10 mM EDTA ("buffer 2")

17 μg HA9 peptide (about 2 μl) ("peptide")

1 μl 2-mercaptoethanol ("βME")

The following four test mixtures were prepared, in each case made up to10 μl with water:

1) cys bridge+buffer 2+βME

2) cys bridge+buffer 2

3) cys bridge+buffer 2+βME+peptide

4) cys bridge+buffer 2+peptide

The mixtures were incubated for 1 hour at room temperature. There wasthen added 1 μl of a mixture of 0.37M sodium tetrathionate and 1.6Msodium sulphite (which had been freshly prepared in accordance with themethod of Morehead et al., Biochem., 23, 1984, 2500). The mixtures wereleft overnight at room temperature.

The mixtures were analysed using a PAGE Schagger System (Schagger etal., 1987, Anal. Biochem., 166, 368-379), blotted onto nitrocellulosepaper using a Bio-Rad Western blotting apparatus, with a transfer bufferof 39 mM glycine, 48 mM Tris, 0.1% (w/v) sodium dodecyl sulphate (SDS)and 20% methanol for a transfer time of 1 hour at 450 mA.

The blots were washed with phosphate buffered saline (PBS) pH 7.6containing Tween 20 (polyoxyethylene sorbitan monolaurate--3 ml perliter PBS) to equilibrate. They were then incubated for 1 hour at 37° C.with 35 ml PBS-Tween plus 0.5% (w/v) bovine serum albumin (BSA), washed6×5 min. with 200 ml PBS-Tween and subsequently incubated overnight at4° C. with 35 ml PBS-Tween+0.5% (w/v) BSA together with 100 μl mouseanti-HA9 monoclonal antibody (obtained from Balcore Co., Berkley,U.S.A.). There then followed washing with PBS-Tween (6×5 min.--200 ml)and incubation for half an hour with 35 ml PBS-Tween+0.5% (w/v) BSAtogether with 50 μl goat anti-mouse IgG horseradish peroxidase (HRP)conjugate. After further washing (6×6 min.--200 ml PBS-Tween), the gelwas excited by luminol Western blotting reagents (Amersham) andvisualised.

The results showed that only a single band in the lane corresponding tosample number 4 cross-reacted with the anti-HA9 antibody. This is theexpected result, samples 1-3 being negative controls. Thus it ispossible to couple linear peptide fragments to cys 14 capsids usingthese methods.

G) Covalent Cross-Linking of cys 14 Modified Coat Protein to an Enzymeor Targeting Ligand Protein

The purified cys 14 modified capsids described in E) above werecovalently linked via a maleimide group to the enzyme horseradishperoxidase (HRP) as follows:

HRP-maleimide conjugate (Pierce Europe BV, Holland), cys 14 modifiedcapsids and βME were used to make the following mixtures, each of whichwas made up to 100 μl with 100 mM NaPi, pH 7.2:

1) 20 μg HRP-maleimide plus 1 μl βME

2) 20 μg cys 14 modified capsids plus 1 μl βME

3) 20 μg cys 14 modified capsids plus 20 μg HRP-maleimide

Sample 3) was left for 1 hour at room temperature and then βME added toquench any remaining thiols. Samples 1-3 were then fractionated by HPLCgel filtration chromatography on PW 3000, 2×30 cm columns, in 100 mMNaPi, pH 7.2 at a flow rate of 0.5 ml/min. Fractions (1 min.--0.5 ml) ofthe eluate were then assayed for HRP activity using the commerciallyavailable kit (ABTS reagent, Pierce), enzyme activity being estimated byobserving the increased absorbance of solutions at 410 nm. The datashowed a significant increase over background levels in fractionscorresponding to the OD₂₈₀ peak of cys 14 assembled material of sample3.

H: Coupling of SATA-modified MS2 WT CP to maleimide-activated HRP.

Maleimide-activated HRP was purchased from Pierce at a concentration of1 mg/ml. 1 ml of SATA-modified MS2 was reacted with 0.1 mg HRP at RT for6 h and then transferred to 4° C. for storage. Samples passed over HPLCgel filtration columns were analysed to determine the efficiency of theconjugation by both enzyme assay for HRP and Western Blots with anti-CPantibodies, which were consistent with formation of a covalentcross-link between the capsid and HRP.

I: Coupling of SATA-modified MS2 WT CP to maleimide-activatedtransferrin (TF).

MS2 was modified with SATA using the same methods as described above. TFwas then activated for reaction as follows: 1 mg of transferrin (TF) wasdissolved in 0.5 ml of 0.05M PB, pH7.5, and 0.5 mg of Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) dissolved in30 μl of PB, and then the two solutions were mixed and kept at 37° C.for 30 min with gentle shaking. The precipitates were removed byspinning at 3 k rpm for 10 min at 4° C. and the supernatant passed overa NAP-25 column equilibrated in 0.05M PB.

Conjugates were made as follows: 1 ml of MS2-SATA (0.75 mg/ml) was addedto 1 ml of maleimide-activated TF (0.44 mg/ml) in a solution flushedwith nitrogen gas and kept at RT for 6 h then stored at 4° C. Conjugatesappeared stable over a period of at least one month at 4° C. The sampleswere analysed by HPLC chromatography and Western blotting with bothanti-CP and anti-transferrin antibodies, as described above. The resultsconfirmed formation of covalent cross-links between the capsids andtransferrin.

H) Cell Entry of MS-2

Wild type recombinant MS-2 RNA-empty capsids prepared as described in A)above were allowed to react with rabbit polyclonal anti-MS-2 serum andthen incubated for one hour with mouse macrophages. The macrophagesnormally function by binding to Fc portions of immuno-complexes via acell surface receptor which is then endocytosed. The experiment thussought to demonstrate cell entry of MS-2 particles via endocytosis. Theresults were analysed by embedding the macrophage cells in a plasticblock followed by thin sectioning and various staining procedures. Theresults are shown in FIGS. 12 to 15. FIGS. 12 and 13 show macrophagesections negatively stained. 300 Å particles (MS-2 capsids) can clearlybe seen in essentially all the sections viewed. FIGS. 14 and 15 showsimilar sections which have been treated with anti-rabbit peroxidasewhich then stains the anti-MS-2 antibodies (black dots in photograph).Both 300 Å and larger aggregates are heavily stained and in the lastpanel the staining is clearly dispersing within the cell as would bepredicted following dissociating of the complex in the lysosome.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS:  3                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 19                                                              <212> TYPE: RNA                                                               <213> ORGANISM: Phage MS-2                                                    - <400> SEQUENCE: 1                                                           # 19               ugu                                                        - <210> SEQ ID NO 2                                                           <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:  PhageMATION: Description of Artificial                            #conjugated toranslational operator RNA                                       #against Tatsense oligonucleotide directed                                    #       mRNA                                                                  - <400> SEQUENCE: 2                                                           #    40            ugut acctcggtca tctaggattg                                 - <210> SEQ ID NO 3                                                           <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Human influenza virus                                         - <400> SEQUENCE: 3                                                           - Tyr Pro Tyr Asp Val Pro Asp Tyr Ala                                           1               5                                                           __________________________________________________________________________

What is claimed is:
 1. A delivery system comprising a capsid formed froma coat protein of a bacteriophage selected from the group consisting ofMS-2, R17, fr, GA, Qβ, and SP and a foreign moiety enclosed in thecapsid, wherein the foreign moiety is of a size sufficiently small to beenclosed in the capsid and wherein the foreign moiety is linked to a RNAsequence comprising a translational operator of the bacteriophage, whichtranslational operator binds to the coat protein during formation of thecapsid.
 2. The delivery system of claim 1, wherein the bacteriophage isMS-2 or R17 and the RNA sequence comprises the sequence of SEQ ID NO:1.3. The delivery system of claim 2, wherein the base position at 11 ofthe sequence of SEQ ID NO:1 is replaced with cytidine.
 4. The deliverysystem of claim 1, wherein the foreign moiety is linked directly to theRNA sequence.
 5. The delivery system of claim 1, wherein the foreignmoiety is linked to the RNA sequence through a spacer moiety.
 6. Thedelivery system of claim 1, wherein the N-terminal protuberant β-hairpinof the coat protein of the capsid has been modified to provide a sitesuitable for attachment thereto of a targeting moiety.
 7. The deliverysystem of claim 6, wherein the site is a cysteine residue.
 8. Thedelivery system of claim 6, wherein a targeting moiety is directlyattached to the coat protein or linked to the coat protein through aspacer moiety.
 9. The delivery system of claim 8, wherein the targetingmoiety comprises galactose.
 10. The delivery system of claim 1, whereinthe foreign moiety is selected from the group consisting of a gene, genefragment, ribozyme, anti-sense oligonucleotide, cytotoxic agent andchemotherapeutic agent.
 11. The delivery system of claim 1, wherein thebacteriophage is GA.
 12. The delivery system of claim 1, wherein thebacteriophage is Qβ.
 13. The delivery system of claim 1, wherein thebacteriophage is SP.
 14. The delivery system of claim 1, wherein thebacteriophage is fr.
 15. A method of preparing a delivery system ofclaim 1, comprising:providing the foreign moiety; linking the foreignmoiety to the RNA sequence to produce the foreign moiety linked to theRNA sequence; providing the capsid; and incorporating into the capsidthe foreign moiety linked to the RNA sequence, such that the capsidencloses the foreign moiety.
 16. The method of claim 15, wherein theforeign moiety linked to the RNA sequence is incorporated into thecapsid by disassembly of the capsid at acid pH in the presence of theforeign moiety linked to the RNA sequence followed by reassembly of thecapsid at an increased pH.